Bone cell culture: Osteoblasts, osteoclasts and osteocytes - Anna Teti
Bone cell culture: Osteoblasts, osteoclasts and osteocytes - Anna Teti
Bone cell culture: Osteoblasts, osteoclasts and osteocytes - Anna Teti
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ECTS PhD Training course<br />
<strong>Bone</strong> Cell Culture: <strong>Osteoblasts</strong>,<br />
Osteoclasts <strong>and</strong> Osteocytes<br />
<strong>Anna</strong> <strong>Teti</strong><br />
Department of Experimental Medicine<br />
University of L’Aquila<br />
L’Aquila, Italy<br />
teti@univaq.it
L’Aquila
L’Aquila
Gran Sasso d’Italia
L’Aquila = The Eagle
<strong>Bone</strong> Cell Technology
<strong>Bone</strong> Cells
<strong>Bone</strong> Cell Array
Osteoblast Isolation<br />
• Wong G, Cohn DV. Separation of<br />
parathyroid hormone <strong>and</strong> calcitoninsensitive<br />
<strong>cell</strong>s from non-responsive bone<br />
<strong>cell</strong>s. Nature 1974 252:713-715.<br />
• Robey, P. <strong>and</strong> Termine, J. D. Human<br />
bone <strong>cell</strong>s in vitro. Calcif Tissue Int 1985<br />
37:453-460.
Sequential Trypsin-Collagenase<br />
Digestion<br />
Human<br />
Mouse
Osteoblast Morphology<br />
<strong>Osteoblasts</strong><br />
Fibroblasts
Osteoblast Markers<br />
Alkaline phosphatase<br />
Cohen, 2006
Cultured <strong>Osteoblasts</strong><br />
Phase contrast<br />
Alkaline Phosphatase<br />
Collagen I<br />
Mineralization
Ascorbic acid<br />
β-glycerophosphate<br />
Mineralized Nodule
Mineralized Nodule<br />
Mineralized<br />
matrix<br />
Osteoblast<br />
Osteocyte
Mesenchymal Stem Cells
Mesenchymal Stem Cells<br />
• Adipogenic<br />
• Insulin<br />
• Dexamethasone<br />
• Isobutyl-methylxanthine<br />
•Osteogenic<br />
•BMP2<br />
• Ascorbic acid<br />
• β-glycerophosphate<br />
•Chondrogenic<br />
• Insulin<br />
• Ascorbic acid<br />
•TGFβ1
Trans-differentiation from<br />
Adipocytes
Trans-differentiation into<br />
Adipocytes
Circulating (Pre)-<strong>Osteoblasts</strong><br />
Sorted by<br />
MACS (anti-OC)<br />
Separated by FACS<br />
Eghbali-Fatourechi et al., 2007
Model for Circulating<br />
(Pre)-<strong>Osteoblasts</strong><br />
Eghbali-Fatourechi et al., 2007
Osteocytes
Ossicles of the Ear
Osteocytes
Step procedure<br />
1<br />
2<br />
3<br />
4<br />
5<br />
6<br />
7<br />
8<br />
9<br />
Soft tissues <strong>and</strong> periosteum are removed from the long bones of 3-day-old rats.<br />
<strong>Bone</strong>s are dipped into 70% ethanol for 15 s to kill most of the <strong>cell</strong>s in the<br />
superficial layer.<br />
Epiphyses are cut off, shaft is cut longitudinally, <strong>and</strong> bone marrow is removed by<br />
scraping <strong>and</strong> extensive washing. <strong>Bone</strong> pieces are washed with PBS (5×3 min<br />
each).<br />
Pieces of bone are incubated in collagenase solution at 37°C for 20 min, four<br />
times (fractions 1–4).<br />
Supernatants are collected at each time <strong>and</strong> centrifuged at 200g for 5 min. Pellets<br />
are suspended in <strong>cell</strong> <strong>culture</strong> medium for <strong>cell</strong> <strong>culture</strong>. <strong>Bone</strong> pieces are rinsed<br />
twice with Hanks' balanced salt solution (HBSS), <strong>and</strong> twice with PBS.<br />
Residual bone pieces are treated with 5 mM EDTA in PBS containing 0.1% BSA at<br />
37°C for 20 min. Step 5 is repeated (fraction 5).<br />
<strong>Bone</strong> pieces are treated with collagenase solution at 37°C for 20 min. Step 5 is<br />
repeated (fraction 6).<br />
<strong>Bone</strong> pieces are cut into smaller pieces, treated with EDTA at 37°C for 20 min.<br />
Step 5 is repeated (fraction 7).<br />
Residual bone pieces are washed with HBSS <strong>and</strong> PBS <strong>and</strong> treated with<br />
collagenase solution at 37°C for 20 min. Step 5 is repeated (fraction 8).
Osteocytes
Cell Types in the Various Cell<br />
Fractions<br />
Fraction<br />
1+2<br />
3+4<br />
5<br />
6<br />
7<br />
8<br />
Fibroblast-like<br />
<strong>cell</strong>s a<br />
52.5%<br />
28.3%<br />
26.9%<br />
22.6%<br />
10.7%<br />
10.6%<br />
Osteoblast-like<br />
<strong>cell</strong>s b<br />
36.8%<br />
50.3%<br />
29.1%<br />
18.9%<br />
12.8%<br />
11.5%<br />
a<br />
ALP-negative elongated spindle-shaped <strong>cell</strong>s<br />
b<br />
ALP-positive <strong>and</strong> cuboidal in shape<br />
c<br />
ALP-negative (or only weakly positive) stellate <strong>cell</strong>s<br />
Osteocyte-like<br />
<strong>cell</strong>s c<br />
10.7%<br />
21.4%<br />
44.0%<br />
58.5%<br />
76.5%<br />
77.9%<br />
Gu et al., 2006
Osteocyte Phenotype<br />
Fibroblasts<br />
<strong>Osteoblasts</strong><br />
Osteocyte<br />
ALP<br />
Osteocalcin<br />
Gui et al., 2006
Osteocyte Phenotype<br />
Connexin 43<br />
Gu et al., 2006
Role of Osteocytes
Osteoclasts
<strong>Teti</strong> <strong>and</strong> the Osteoclasts…
<strong>Teti</strong> <strong>and</strong> the Osteoclasts…<br />
….30 years later
Chicken Osteoclasts
Chicken Osteoclasts
Podosomes
Rat Osteoclasts
Pit Assay
Osteoclast Markers<br />
TRAcP Actin ring VNR<br />
•CTR<br />
• Cathepsin K<br />
• MMP9<br />
•Pits
Mechanisms of <strong>Bone</strong> Resorption<br />
RANKL<br />
RANK<br />
H 2<br />
O + CO 2<br />
CAII<br />
H + + HCO3-<br />
HCO3-<br />
PLEKHM1<br />
OSTM1<br />
CL -<br />
MMP9<br />
CatK<br />
ClCN7<br />
V-ATPase<br />
CL -<br />
H +<br />
Resorbing Lacuna
Pit Assay<br />
Mulari et al., 2005
Resorption Pits<br />
Sobacchi et al., 2007
Hydroxyapatite Resorption
Collagen Degradation Assay
Osteoclast Ontogeny<br />
Macrophage<br />
Monocyte<br />
Osteoblast<br />
Haematopoietic<br />
precursors<br />
Myeloid <strong>cell</strong><br />
Osteoclast<br />
Polarized<br />
osteoclast<br />
Pre-osteoclast
Osteoblast-Osteoclast Co-<strong>culture</strong><br />
Treatment with 1,25(OH) 2<br />
VitaminD 3
Osteoclast Ontogeny<br />
Macrophage<br />
Monocyte<br />
Osteoblast<br />
Haematopoietic<br />
precursors<br />
Myeloid <strong>cell</strong><br />
RANKL M-CFS<br />
RANK c-fms<br />
Osteoclast<br />
Polarized<br />
osteoclast<br />
Pre-osteoclast
M-CSF <strong>and</strong> RANKL<br />
RANKL<br />
M-CSF<br />
RANK<br />
c-Fms<br />
Osteoblast<br />
Osteoclast
Purified Osteoclasts<br />
Human Osteoclasts from Peripheral Blood Monocyes
Osteoclast Signalling<br />
Baron, 2004<br />
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